195 RELATIVE mRNA EXPRESSION OF 4 CANDIDATES IN LAMB OOCYTES SELECTED BY BRILLIANT CRESYL BLUE STAINING

Abstract

Brilliant cresyl blue (BCB) staining determines the activity of glucose-6-phosphate dehydrogenase (G6PDH), an enzyme in which activity decreases as the oocytes reach their growth phase. We have previously shown in lamb the effectiveness of this stain in selecting the largest and most competent oocytes to develop up to the blastocyst stage (Catala et al. 2011 Reproduction 142, 517–527). The aim of this study was to analyse the expression of 4 genes related to oocyte quality in BCB-selected oocytes. The 4 genes analyzed in this study were selected after a literature review in which they were associated with a better oocyte quality or embryo development; 2 of these genes were related to cell cycle: nuclear auto-antigenic sperm protein (NASP) and histone H2A (H2A.Z), and 2 genes with enzymatic functions: peroxiredoxin (PRDX1) and elongation factor-A1 (EEF1A1). Oocytes were recovered after slicing the surface of lamb ovaries (3 to 5 months old) obtained from a local slaughterhouse. Oocytes with more than 3 compact cumulus layers and homogenic cytoplasm were selected and exposed to 13 µM BCB during 1 h before IVM and classified according to oocyte coloration: oocytes with blue cytoplasm or grown oocytes (BCB+) and oocytes not coloured or growing oocytes (BCB–). Cumulus–oocyte complexes were then matured in conventional TCM199 medium supplemented with 10% fetal bovine serum and hormones during 24 h in a controlled atmosphere. Groups of 15 denuded oocytes (3 replicates) were taken before (0 h) and after maturation (24 h) and stored at –80°C in 100 mL of Trizol until use. After RNA extraction following manufacturer’s protocol (Promega, Madison, WI, USA), reverse transcription was performed by extended cDNA using Oligo (dT) primers during 5 min at 70°C and 1 h at 65°C using superscript III (200 U mM^–1; Invitrogen). Relative qualitative PCR analyses were performed in duplicate using SYBR Green Fluorophore kit (Bio-Rad, Hercules, CA, USA). The specificity of each PCR product was determined by a melting curve analysis and the amplicon size determination by an agarose gel. A standard curve was also included, consisting of corresponding plasmid DNA fragments from 1 pg to 0.1 fg, purified with QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA). Correlation coefficients and PCR efficiencies were considered between 85 and 100%. Finally, the results for mRNA were normalized according to the relative concentration of the internal and external gene (luciferase and 18S, respectively). The statistical analysis was performed by One-way ANOVA in GraphPad Prism v 3 (GraphPad Software, San Diego, CA, USA). The analysis of gene expression showed no differences in relation to BCB classification. The only difference we found is a significant decrease (P ≤ 0.05) in the RE of PRDX observed in matured oocytes compared with immature ones. In conclusion, the expression of the mRNA expression of NASP, H2A.Z, PRDX1, and EEF1A1 were not affected by oocyte quality.